Conserved residues of the C-terminal p16 domain of primase are involved in modulating the activity of the bacterial primosome

Kiran Chintakayala, Marilynn A Larson, Mark A Griep, Steven Heye Hinrichs, Panos Soultanas

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

The bacterial primosome comprises the replicative homo-hexameric ring helicase DnaB and the primase DnaG. It is an integral component of the replisome as it unwinds the parental DNA duplex to allow progression of the replication fork, synthesizes the initiation primers at the replication origin, oriC, and the primers required for Okazaki fragment synthesis during lagging strand replication. The interaction between the two component proteins is mediated by a distinct C-terminal domain (p16) of the primase. Both proteins mutually regulate each other's activities and a putative network of conserved residues has been proposed to mediate these effects. We have targeted 10 residues from this network. To investigate the functional contributions of these residues to the primase, ATPase and helicase activities of the primosome, we have used site-directed mutagenesis and in vitro functional assays. Five of these residues (E464, H494, R495, Y548 and R555) exhibited some functional significance while the remaining five (E483, R484, E506, D512 and E530) exhibited no effects. E464 participates in functional modulation of the primase activity, whereas H494, R495 and R555 participate in allosteric functional modulation of the ATPase and/or helicase activities. Y548 contributes directly to the structural interaction with DnaB.

Original languageEnglish (US)
Pages (from-to)360-371
Number of pages12
JournalMolecular Microbiology
Volume68
Issue number2
DOIs
StatePublished - Apr 1 2008

Fingerprint

DNA Primase
Adenosine Triphosphatases
DnaB Helicases
Replication Origin
Site-Directed Mutagenesis
Proteins
DNA

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

Conserved residues of the C-terminal p16 domain of primase are involved in modulating the activity of the bacterial primosome. / Chintakayala, Kiran; Larson, Marilynn A; Griep, Mark A; Hinrichs, Steven Heye; Soultanas, Panos.

In: Molecular Microbiology, Vol. 68, No. 2, 01.04.2008, p. 360-371.

Research output: Contribution to journalArticle

@article{07df3a2728bb453392fd1948d218ba6d,
title = "Conserved residues of the C-terminal p16 domain of primase are involved in modulating the activity of the bacterial primosome",
abstract = "The bacterial primosome comprises the replicative homo-hexameric ring helicase DnaB and the primase DnaG. It is an integral component of the replisome as it unwinds the parental DNA duplex to allow progression of the replication fork, synthesizes the initiation primers at the replication origin, oriC, and the primers required for Okazaki fragment synthesis during lagging strand replication. The interaction between the two component proteins is mediated by a distinct C-terminal domain (p16) of the primase. Both proteins mutually regulate each other's activities and a putative network of conserved residues has been proposed to mediate these effects. We have targeted 10 residues from this network. To investigate the functional contributions of these residues to the primase, ATPase and helicase activities of the primosome, we have used site-directed mutagenesis and in vitro functional assays. Five of these residues (E464, H494, R495, Y548 and R555) exhibited some functional significance while the remaining five (E483, R484, E506, D512 and E530) exhibited no effects. E464 participates in functional modulation of the primase activity, whereas H494, R495 and R555 participate in allosteric functional modulation of the ATPase and/or helicase activities. Y548 contributes directly to the structural interaction with DnaB.",
author = "Kiran Chintakayala and Larson, {Marilynn A} and Griep, {Mark A} and Hinrichs, {Steven Heye} and Panos Soultanas",
year = "2008",
month = "4",
day = "1",
doi = "10.1111/j.1365-2958.2008.06155.x",
language = "English (US)",
volume = "68",
pages = "360--371",
journal = "Molecular Microbiology",
issn = "0950-382X",
publisher = "Wiley-Blackwell",
number = "2",

}

TY - JOUR

T1 - Conserved residues of the C-terminal p16 domain of primase are involved in modulating the activity of the bacterial primosome

AU - Chintakayala, Kiran

AU - Larson, Marilynn A

AU - Griep, Mark A

AU - Hinrichs, Steven Heye

AU - Soultanas, Panos

PY - 2008/4/1

Y1 - 2008/4/1

N2 - The bacterial primosome comprises the replicative homo-hexameric ring helicase DnaB and the primase DnaG. It is an integral component of the replisome as it unwinds the parental DNA duplex to allow progression of the replication fork, synthesizes the initiation primers at the replication origin, oriC, and the primers required for Okazaki fragment synthesis during lagging strand replication. The interaction between the two component proteins is mediated by a distinct C-terminal domain (p16) of the primase. Both proteins mutually regulate each other's activities and a putative network of conserved residues has been proposed to mediate these effects. We have targeted 10 residues from this network. To investigate the functional contributions of these residues to the primase, ATPase and helicase activities of the primosome, we have used site-directed mutagenesis and in vitro functional assays. Five of these residues (E464, H494, R495, Y548 and R555) exhibited some functional significance while the remaining five (E483, R484, E506, D512 and E530) exhibited no effects. E464 participates in functional modulation of the primase activity, whereas H494, R495 and R555 participate in allosteric functional modulation of the ATPase and/or helicase activities. Y548 contributes directly to the structural interaction with DnaB.

AB - The bacterial primosome comprises the replicative homo-hexameric ring helicase DnaB and the primase DnaG. It is an integral component of the replisome as it unwinds the parental DNA duplex to allow progression of the replication fork, synthesizes the initiation primers at the replication origin, oriC, and the primers required for Okazaki fragment synthesis during lagging strand replication. The interaction between the two component proteins is mediated by a distinct C-terminal domain (p16) of the primase. Both proteins mutually regulate each other's activities and a putative network of conserved residues has been proposed to mediate these effects. We have targeted 10 residues from this network. To investigate the functional contributions of these residues to the primase, ATPase and helicase activities of the primosome, we have used site-directed mutagenesis and in vitro functional assays. Five of these residues (E464, H494, R495, Y548 and R555) exhibited some functional significance while the remaining five (E483, R484, E506, D512 and E530) exhibited no effects. E464 participates in functional modulation of the primase activity, whereas H494, R495 and R555 participate in allosteric functional modulation of the ATPase and/or helicase activities. Y548 contributes directly to the structural interaction with DnaB.

UR - http://www.scopus.com/inward/record.url?scp=41049104425&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=41049104425&partnerID=8YFLogxK

U2 - 10.1111/j.1365-2958.2008.06155.x

DO - 10.1111/j.1365-2958.2008.06155.x

M3 - Article

C2 - 18366438

AN - SCOPUS:41049104425

VL - 68

SP - 360

EP - 371

JO - Molecular Microbiology

JF - Molecular Microbiology

SN - 0950-382X

IS - 2

ER -